The Pig-tailed Macaque Rectal Model

The Pig-tailed Macaque Rectal Model Microflora and Chlamydial Infection DOROTHY L. PATTON, PHD,* YVONNE T. COSGROVE-SWEENEY,* LORNA K. RABE,† AND SHARON L. HILLIER, PHD† From the *Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington; and the † Department of Obstetrics and Gynecology and Reproductive Sciences, University of Pittsburgh, MageeWomens Hospital, Pittsburgh, Pennsylvania Background: A topical microbicide should protect against acquisition of sexually transmitted infection during both vaginal and rectal intercourse. The rectal microflora of the Macaca nemestrina (pig-tailed macaque) and humans were examined, as well as the histopathology of rectal tissues. In a subset of macaques, a human rectal isolate of Chlamydia trachomatis was inoculated into the rectum to establish rectal chlamydial infection. Goal: To evaluate the comparability of the pig-tailed macaque rectal model with humans. Study Design: Rectal swabs were collected for microbiologic analysis to characterize normal microflora in pig-tailed macaques and humans. Subsequently, 10 macaques received a rectal inoculation with C trachomatis, serovar D, prepared from a clinical rectal isolate. Results: The rectal microflora of pig-tailed macaques (n 5 80) were found to be comparable with the rectal flora of humans (n 5 40). The prevalence of Lactobacillus in the rectum was higher in the macaques than in humans. Coliform and Enterococcus were decreased in the macaques, as compared with those of humans. In 9 of 10 macaques, rectal chlamydial infection was confirmed by culture or ligase chain reaction on days 2, 7, and 14 after inoculation. The test results were positive for rectal chlamydial infection by ligase chain reaction only for the remaining animal on day 14 after inoculation. Conclusions: The findings demonstrate that the rectal environment of the pig-tailed macaque is a useful model for further evaluation of newly developed topical microbicides for rectal use. Furthermore, such products can be evaluated for protection against rectal chlamydial infection in this model. ranges from 5% or more in the prime reproductive age group2 to more than 20% in some sexually transmitted disease clinics. Those most prone to infection are unmarried young women (i.e., younger than 22 years) with multiple sex partners, who frequently are asymptomatic. Several studies have shown that C trachomatis can be isolated from the rectums of approximately 5% of women and 15% of homosexual men reporting to sexually transmitted disease clinics.3– 6 Healthcare costs attributed to chlamydial infections and their sequelae reached $4.2 billion in 1990.7 Given the economic impact of chlamydial infection, there is a need to evaluate topical microbicides applied either vaginally or rectally to reduce its transmission. Anorectal infections caused by C trachomatis are not uncommon, yet animal models for the study of such infections are scarce. Over the past 15 years, the female pigtailed macaque (Macaca nemestrina) has been used to investigate the pathogenesis of experimentally induced C trachomatis cervicitis and salpingitis/pelvic inflammatory disease.8 The macaque was chosen for these studies for several reasons: It has a 28- to 30-day menstrual cycle; its anatomy and physiology are well characterized and similar to those of humans; and it is naturally susceptible to the human biovars of C trachomatis. In this study, we have extended the usefulness of the C trachomatis cervicitis/salpingitis model to evaluate topical microbicides candidates. We established that the vaginal flora and epithelial tissues are remarkably similar to those in women.9 We have used this model to study the effects of single and repeated applications of spermicides and microbicides on the vaginal microflora and epithelium.10 Further, IN THE UNITED STATES, Chlamydia trachomatis is one of the most common sexually transmitted pathogens, with an estimated 4 million new cases of infection each year.1 Approximately 2.5 million infections occur in women each year. Among women, chlamydia has a prevalence that Supported in part by grants P01-AI-39061 from the Public Health Services and RR 00166 of the Washington Regional Primate Research Center. Human studies were supported in part by N01-AI-75326. Correspondence: Dorothy Patton, PhD, Department of Obstetrics and Gynecology, University of Washington, Box 356460, Seattle, WA 981956460. E-mail: [email protected] Received for publication August 9, 2000, revised December 1, 2000, and accepted December 12, 2000. 363 364 PATTON ET AL we have investigated whether these products are chlamydiacidal in the monkey model.11,12 In the current study, we first established that the rectal flora and epithelial tissues of the pig-tailed macaque are similar to those in humans. Secondly, we documented that the rectum of the pig-tailed macaque is naturally susceptible to human isolates of rectal C trachomatis. We propose that this extension of the macaque model will be useful for further study investigating the effects of topical microbicides on the rectal microflora and epithelium, and for investigating chlamydiacidal activity in anorectal tissue. Sexually Transmitted Diseases ● July 2001 vaginitis. To assess vaginal flora, vaginal smears were collected from all the women. Histology of Rectal Biopsy Tissue Rectal biopsy specimens were collected from the macaques and compared with archived human tissues obtained from the Department of Pathology at the University of Washington. Routine histology was assessed from formalinfixed paraffin-embedded sections stained with hematoxylin and eosin. Organisms Methods Experimental Animals In this study, 80 (70 female and 10 male) feral- or colony-born sexually mature pig-tailed monkeys (Macaca nemestrina) were used to characterize normal rectal microflora. All the monkeys were housed at the University of Washington’s Regional Primate Research Center. Prior approval for the use of monkeys in this protocol was obtained from the University of Washington’s Animal Care Committee. The animals were handled humanely, and the experiments were performed within NIH Animal Use Guidelines. Rectal swabs were collected and transported in a Port-ACul anaerobic transport tube (Becton-Dickinson, Cockeysville, MD) to the microbiology laboratory at MageeWomens Hospital at Pittsburgh, Pennsylvania, by express mail in Styrofoam packing material within 24 hours of collection. This transport device has been shown to preserve the viability of aerobic and anaerobic bacteria in clinical samples.13 Humans Rectal swabs were collected from 40 healthy, asymptomatic women ages 18 to 30 years in Pittsburgh, Pennsylvania. After written informed consent had been provided, a Dacron swab was inserted into the anorectum of each woman and placed into a culture transport device. The specimens were transported to the laboratory and inoculated with the appropriate culture media on the day of collection. The protocol for collection of the specimens was approved by the MageeWomens Hospital Institutional Review Board. Microbiologic Characterization of Microflora Swabs from all the humans and animals were evaluated for aerobic and anaerobic microorganisms according to microbiologic assays previously described.9 Species belonging to the genera Bacteroides, Porphyromonas, and Prevotella were grouped together as anaerobic gram-negative rods for ease of presentation. None of the women had symptoms of Rectal strain D (MT7226) of C trachomatis was prepared in McCoy cell culture and purified by renografin methylglucamine diatrizoate linear gradient column. The inocula, containing 5 3 107 inclusion-forming units per milliliter, were aliquoted in sucrose-phosphate-glutamate buffer and frozen at 270 °C until used for the study. Inoculation and Specimen Collection The animals were intramuscularly anesthetized with ketamine-xylazine, and 10 animals were inoculated with C trachomatis. Just before sedation, chlamydia was thawed and diluted to 1.0-ml aliquots of 5 3 105 inclusion forming units (IFU) per milliliter each. At each inoculation, 1.0 ml of inoculum was dispensed into the rectal canal with a 1-ml syringe. After inoculation, rectal specimens were collected with Dacron-tipped swabs inserted 3 to 4 cm beyond the anal sphincter on days 2, 7, and 14 for detection of C trachomatis. Chlamydia trachomatis Detection Assays Cell culture. Specimens were cultured on cycloheximide-treated McCoy cells in 96-well microtiter plates14 and stained with a monoclonal antibody specific for C trachomatis serovar D. Ligase chain reaction. The rectal swab specimens were centrifuged, then incubated at 95 to 100 °C for 15 minutes, followed by cooling to 20 °C. Before use in ligase chain reaction (LCR) assays,15 the specimens were either frozen for batch runs or refrigerated to be processed within 48 hours. The specimens subsequently were added to the chlamydia LCR unit dose tubes (provided by Abbott Laboratories, Abbott Park, IL) and then amplified according to the manufacturer’s instructions. Results Validation of the Nonhuman Primate Rectal Model Rectal microflora. The facultative and anaerobic microorganisms recovered from 80 pig-tailed macaques are listed Vol. 28 ● No. 7 MICROFLORA AND CHLAMYDIAL INFECTION TABLE 1. Comparison of Rectal Microflora Between Monkeys and Humans Organism Any Lactobacillus Lactobacillus H2O21 Lactobacillus H2O22 Beta hemolytic streptococci Enterococcus spp. Any viridans streptococcus Viridans streptococci H2O21 Viridans streptococci H2O22 Coagulase negative staphylococcus Coliform Anaerobic positive cocci Black anaerobic GNR* Other anaerobic GNR Macaca nemestrina (n 5 80) % 100 80 54 0 14 72 20 69 29 29 70 90 98 Human (n 5 40) % 68 48 20 33 38 60 25 (n520) 55 (n520) 28 100 98 95 98 *GNR 5 gram-negative rods. in Table 1. The rectal culture results from 40 reproductive-age women are presented for comparison. As shown in Table 1, the microorganisms making up the normal rectal microflora of the pig-tailed macaque is similar to that of the human. Rectal colonization by H2O2-producing lactobacilli was higher in the macaques than in the women (80% versus 48%). There was a relation between vaginal flora pattern and rectal colonization by lactobacilli. In the analysis, H2O2-producing lactobacilli were detected in the rectum specimens of 14 (64%) of 22 women with normal smears, 5 (56%) of 9 women with intermediate smears, and none (0%) of 9 women with Fig. 1. Light microscopy of normal rectal biopsies from the pig-tailed macaque and the human. The tissues comprise approximately 15 layers of stratified squamous epithelium. The lamina propria of the rectal tissue is composed of loose connective tissue with numerous blood vessels (hematoxylin and eosin; original magnification, 340). 365 bacterial vaginosis smears. Therefore, the revalence of rectal lactobacilli observed in women may be dependent on their vaginal microflora and bacterial vaginosis prevalence. Anaerobic gram-positive cocci were found in 70% of the macaques, as compared with 98% of the humans. Interestingly, as shown in Table 1, the levels of coliform and Enterococcus microorganisms in the macaque rectum were markedly lower than in the human rectum. Of the microorganisms studied, only beta hemolytic streptococci were found to be absent from the rectal microflora of the macaques. Rectal histology. The biopsies of the rectal tissues comprised approximately 15 layers of stratified squamous epithelium. The lamina propria of these tissues was composed of loose connective tissue with numerous blood vessels. These findings are consistent with those from human rectal tissues (Figure 1). The stratified squamous epithelium of the rectum has approximately half the number of epithelium layers as the vagina. Detection of Chlamydia trachomatis. Rectal chlamydial infection developed in the 10 macaques inoculated with rectal strain D of C trachomatis (5 3 105 IFU). The cultures and LCR were positive on days 2, 7, and 14 after inoculation (Table 2). One exception, animal 4, was LCR-positive only on day 14 after inoculation, returning to negative status 1 week later. 366 PATTON ET AL TABLE 2. Sexually Transmitted Diseases ● July 2001 Chlamydia trachomatis Detection in the Rectum by Culture and Ligase Chain Reaction (LCR) in 10 Monkeys 1 2 3 4 5 6 7 8 9 10 Day Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR Culture LCR 0 2 7 14 21 2 1 1 1 2 1 1 1 2 2 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 2 2 2 2 2 2 2 1 2 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 2 1 1 2 1 1 1 2 2 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 1 Discussion References Research efforts to develop topical microbicides for intravaginal use for prevention of sexually transmitted infections have been ongoing for nearly a decade. An estimated 333 million new cases of vaginally and rectally transmitted diseases occur in the world each year.16 Clearly, an ideal microbicide would prevent the transmission of both vaginally and rectally acquired sexually transmitted infections. Therefore, topical microbicide products must be tested to ensure that the sexual transmission of chlamydia and other sexually transmitted infections is prevented, and that the rectal environment, including the rectal epithelium, is preserved. Previously, the current authors established the fact that the normal vaginal microflora of the pig-tailed macaque is similar to the vaginal flora of the human. They documented the similarity in histology between the vaginal tissues of macaques and humans, each characterized by approximately 30 layers of epithelium. Subsequently, they used the current macaque model to study the effects of spermicides and microbicides on vaginal microflora, epithelia, and chlamydial infection. In this study they have characterized the rectal microflora and tissues in the macaque, validating the fact that they are similar to those in humans. However, the prevalence rates of individual microorganisms are variable. Additionally, the macaque model’s usefulness in studying rectally acquired chlamydial infection has been established. In this newly expanded model, topical microbicide products can be assessed with regard to their safety and efficacy in preventing C trachomatis infections at both vaginal and rectal sites. The further development of the pig-tailed macaque model to test the effects and effectiveness of microbicides in the female genital tract and rectum is highly significant. It is essential that the activity of currently available topical microbicidal agents be evaluated in animals before their widespread intravaginal or rectal use is recommended in human populations. This advance in model development is a valuable step in bridging the gap between laboratory and clinical studies. 1. Division of STD/HIV Prevention. 1992 Annual Report. Atlanta, GA: Centers for Disease Control and Prevention, 1993. 2. Recommendations for the prevention and management of Chlamydia trachomatis infection 1993. MMWR Morb Mortal Wkly Rep 1993; 115:1145–1148. 3. Wexner SD. Sexually transmitted diseases of the colon, rectum, and anus: the challenge of the nineties. Dis Colon Rectum 1990; 33: 1048 –1062. 4. Jones RB, Rabinovitch RA, Kartz BP, et al. Chlamydia trachomatis in the pharynx and rectum of heterosexual patients at risk for genital infection. Ann Intern Med 1985; 102:757–762. 5. Thompson CI, MacAulay AJ, Smith IW. Chlamydia trachomatis infections in the female rectums. Genitourin Med 1989; 65:269 –273. 6. Christophersen J, Menn’e T, Friis-Moller A, Nielsen JO, Hansted B, Ohlenschloeger J. Sexually transmitted diseases in hetero-, homoand bisexual males in Copenhagen. Dan Med Bull 1988; 35:285– 288. 7. Cates W, Rolfs RT, Aral SO. Sexually transmitted diseases, pelvic inflammatory disease, and infertility: an epidemic update. Epidemiol Rev 1990; 12:199 –220. 8. Patton DL, Lichentenwalner AB. Animal models for the study of chlamydial infections. In: Stephens RS, et al, eds. Chlamydial Infections. San Francisco: International Chlamydia Symposium, 1998: 641– 650. 9. Patton DL, Cosgrove Sweeney Y, Rabe LK, Hillier SL. The vaginal microflora of pig-tailed macaques and the effects of chlorhexidine and benzalkonium on this ecosystem. Sex Transm Dis 1996; 23: 489 – 493. 10. Patton DL, Kidder GG, Cosgrove Sweeney YT, Rabe LK, Hillier SL. Effects of multiple perturbations of benzalkonium chloride and nonoxynol-9 on the vaginal epithelium in the pig-tailed macaque. Am J Obstet Gynecol 1999; 181:1080 –1087. 11. Patton DL, Cosgrove Sweeney YT, McKay TL, Clark AM, Rabe LK, Hillier SL. 0.25% chlorhexidine gluconate gel: a protective topical microbicide. Sex Transm Dis 1998; 25:421– 424. 12. Patton DL, Ganzle Kidder G, Cosgrove Sweeney Y, Rabe LK, Clark AM, Hillier SL. Effects of nonoxynol-9 on vaginal microflora and chlamydial infection in a monkey model. Sex Transm Dis 1996; 23:461– 464. 13. Baron EJ, Strong CA, McTeague M, Vaisanen M-L, Finegold SM. Survival of anaerobes in original specimens transported by overnight mail services. Clin Infect Dis 1995; 29:5174 –5177. 14. Stamm WE, Tam M, Koester M, Cles L. Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluoresceinconjugated monoclonal antibodies. J Clin Microbiol 1983; 17:666 – 668. 15. Dille BJ, Butzen CC, Birkemeyer LG. Amplification of Chlamydia trachomatis DNA by ligase chain reaction. J Clin Microbiol 1993; 31:729 –731. 16. World Health Organization. Sexually Transmitted Diseases (STDs). Fact Sheet N110, March 1996. Available at: http://www.who.int/inffs/en/fact110.html. Accessed: August 2000.